Liposomes have widespread use as carriers for active ingredients. Neutral or negatively charged liposomes are often used for the delivery of small molecule drugs, whereas positively charged (cationic) or the recently introduced class of amphoteric liposomes are mainly used for the delivery of nucleic acids such as plasmids or oligonucleotides. Important examples for cationic liposomes used for the delivery of nucleic acid cargoes include, but are not limited to Semple et al., Nat. Biotech. (2010) 28:172-176; Akinc et al., Nat. Biotech. (2008) 26:561-569; Chien et al., Cancer Gene Ther. (2005) 12:321-328; de Fougerolles, Nat. Rev. Drug Discov. (2007) 6:443-453; Kim et al., Mol. Ther. (2006) 14:343-350; Morrissey, Nat. Biotech. (2005) 23: 1002-1007; Peer, Science (2008) 319: 627-630 and Santel, Gene Ther. (2006) 13: 1222-1234. Application of amphoteric liposomes for the delivery of nucleic acids has been demonstrated in Andreakos et al., Arthritis Rheum. (2009) 60:994-1005.
Amphoteric liposomes belong to the larger family of pH-sensitive liposomes, which further comprise pH-sensitive anionic or cationic liposomes, prototypes of which have been presented in Lai et al., Biochemistry (1985) 24:1654-1661 and Budker et al., Nat. Biotech. (1996) 14:760-764. Unlike the pH-sensitive anionic or cationic liposomes, amphoteric liposomes are complex structures and comprise at least a pair of lipids having complementary charge. WO 02/066012 discloses a key feature of amphoteric liposomes in that these have a stable phase at both low and neutral pH. WO 02/066012 and WO07/107304 describe a method of loading such particles with nucleic acids starting from a low pH.
Hafez, et al. (Biophys. J. 2000, 79(3), 1438-1446) and WO 02/066012 provide some guidance as to how to select lipid mixtures with truly amphoteric properties and more specifically how to determine their isoelectric point and onset of fusion. Neutral lipids can be additional constituents of amphoteric liposomes. The inclusion of one or more such neutral lipids significantly adds to the complexity of the mixture, especially since the individual amounts of all the components may vary. The very high number of possible combinations of lipids represents a practical hurdle towards a more rapid optimisation of amphoteric liposomes. In this regard, WO08/043575 reveals strategies for the optimization of stability, fusogenicity and cellular transfection of amphoteric liposomes, particularly a method of predicting which mixtures of lipids form satisfactorily stable lamellar phases at high and low pH, whilst forming a fusogenic, hexagonal phase at an intermediate pH.
The amphoteric liposomes according to the abovementioned references are potent transfectants of cells. However, it was observed that the function of some of these liposomes could be blocked by the addition of certain sera, thereby potentially limiting the activity of these liposomes for the targeting of certain cells in vivo. This is further illustrated in the Examples presented herein, e.g., Example 3.
The inhibition of the uptake of amphoteric liposomes observed in different sera is apparently opposite to the recently published activation of cationic carrier through complex formation with lipoproteins, in this case ApoE, as demonstrated in Akinc et al., Mol. Ther. (2010) electronic publication on May 11th, ahead of print. DOI: 10.1038/mt.2010.85
A more detailed investigation revealed lipoproteins as mediators of this inhibitory effect. As shown in Example 4 herein, human serum deficient of lipoproteins is no longer able to inhibit the uptake of liposomes as indicated by the functional delivery of siRNA to the challenged cells. The inventors have now surprisingly and unexpectedly found that certain species of cationic imino lipids in combination with anionic lipids having a carboxyl or phosphate moiety in their polar head groups are particularly advantageous in maintaining transfection activity in the presence of serum. Frequently, a particular advantage was observed when the lipid assemblies or liposomes created from said lipid mixtures were formulated according to the method described herein and in WO08/043575.